白念珠菌新型耐药基因MXR1的敲除与鉴定

目的 构建用于白念珠菌MXR1基因敲除的载体质粒,并通过Ura-Blaster策略敲除MXR1两条等位基因.方法 分别扩增白念珠菌MXR1基因ORF两侧上下游的片段,通过酶切与连接反应,将上下游片段分别插入到p5921质粒的hisG-URA 3-hisG盒两端,从而形成MXR1敲除载体质粒pUC-MXR1-URA3.通过Ura-Blaster策略将载体质粒转染到白念珠菌RM 1000内,并采用PCR和Southern-blot杂交方法鉴定各步转染、复筛所得的阳性克隆.结果 成功获得MXR1基因缺失的菌株.结论 MXR1基因缺失菌株的构建,有助于深入研究白念珠菌耐药机制.     

白念珠菌V-ATPase研究进展

白念珠菌是临床最常见的条件致病真菌,V-ATPase是真核生物中高度保守的质子泵转运复合物,可以维持液泡和细胞质pH稳态.近年来,V-ATPase作为一个抗白念珠菌感染的潜在靶点为人们所关注,本文就V-ATPase抑制剂、相关的其他靶点及抑制剂筛选等方面的研究进展作一综述.     

HOG1基因对白念珠菌超微结构的影响

目的探讨HOG1基因对白念珠菌超微结构的影响。方法设置实验组、HOG21组(hog1/hog1双等位基因缺陷株);对照组、WT组(标准株),分别在扫描电镜及透射电镜下观察两组菌株细胞的超微结构。结果扫描电镜下观察两组细胞均呈圆形或椭圆形,呈多边出芽繁殖的生长方式。但HOG1基因缺陷株细胞表面粗糙、凹凸不平,出芽数目比标准株少;标准株细胞表面光滑,出芽数量较多,可见"花瓣样"结构的芽痕;透射电镜下HOG1基因缺陷株细胞壁结构不完整,电子透明层厚薄不一,部分细胞可见棉絮状电子致密外层局灶性缺失、细胞膜外凸、不连续以及细胞膜周围见囊泡聚集等现象。标准株细胞壁各层结构完整。结论HOG1基因对白念珠菌细胞壁结构具有一定影响。     

白念珠菌菌相转换基因HYR1血清相关启动子活性的研究

目的 探讨血清对白念珠菌菌相转换特异基因HYR1上游启动子活性的影响.方法 在白念珠菌菌相转换基因HYR1上游(-1.8kb～ 43bp)中,取长度不等的片段与LacZ(载体PNG17)报告基因构成重组体,转化酵母菌EGY48,探讨血清对HYR1启动子活性的影响.结果 -600～-400bp区域存在激活启动活性,其他区域未见血清相关的调控元件.结论 在HYR1基因上游-600～-400bp间存在与血清相关的基因调控元件,该区域含有与血清相关的启动转录活性.     

白念珠菌新型耐药基因IPF14744敲除质粒的构建

目的 构建用于白念珠菌IPF 14744基因敲除的载体质粒.方法 分别扩增白念珠菌IPF 14744基因ORF两侧上下游的片段,通过酶切与连接反应,将上下游片段分别插入到p5921质粒的hisG-URA3-hisG盒两端,从而形成IPF 14744敲除载体质粒pUC-14744- URA 3.结果 成功获得IPF 14744基因敲除载体质粒.结论 所获得的质粒pUC-14744- URA3可用于白念珠菌IPF 14744基因的敲除.     

The Candida albicans UBI3 gene encoding a hybrid ubiquitin fusion protein involved in ribosome biogenesis is essential for growth

We have constructed a conditional null mutant Candida albicans strain for the UBI3 gene which encodes a ubiquitin fusion protein involved in ribosome biogenesis. A one-step gene disruption procedure, using the plasmid pCaDis, was designed to place the second copy of the UBI3 gene under the control of the tightly regulated MET3 promoter in a C. albicans heterozygous strain (UBI3/Deltaubi3::hisG), previously isolated in the first step of the ura-blaster protocol. Analysis of the conditional null mutant in repressing and inducing conditions indicates that UBI3 is an essential gene whose expression is required for growth of C. albicans.     

白色念珠菌CRK1基因的敲除及其功能研究

在筛选白色念珠菌促分裂原活化蛋白激酶（ＭＡＰＫ）相关蛋白激酶基因的过程中,得到了ＣＲＫ１（ＣＤＣ２－ｒｅｌａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ １）基因。利用同源重组原理,敲除了ＣＲＫ１双拷贝基因片段,构建了ＣＲＫ１纯合缺失株,发现ＣＲＫ１基因的缺失影响到白色念珠菌的生长速度,絮凝生长和形态发生。     

Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.     

CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响

目的 研究CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响.方法 显微镜下观察,计算菌丝形成率,比较CRK1基因缺失菌（Δcrk1菌）及标准菌SC5314形成菌丝的能力;建立肠黏膜模型,计算黏附率,评价CRK1基因缺失对白念珠菌黏附的影响;MTT法及结晶紫法（CV）评价CRK1基因缺失对白念珠菌生物被膜形成的影响.结果 与SC5314相比,Δcrk1菌分别在10％胎牛血清和RPMI-1640培养条件下形成菌丝能力均较弱,两者之间有统计学差异;Δcrk1菌在60、90、120 min时对肠黏膜的黏附数明显少于标准菌SC5314,两者之间有统计学差异;通过MTT法、结晶紫法两种方法证实了,在经48 h培养后,Δcrk1菌与其标准菌SC5314相比,形成生物膜的能力弱,两者之间差异有统计学差异.结论 CRK 1基因缺失影响白念珠菌菌丝二态性的转化,进而影响黏附力和生物被膜的形成.     

Rapid PCR-based test for identifying Candida albicans by using primers derived from the pH-regulated KER1 gene

A PCR-based method in combination with a simple, reliable and inexpensive DNA extraction procedure for rapid detection of Candida albicans clinical isolates is described here. The extraction protocol is based on a combination of chemical (NaOH and detergents) and physical (boiling) treatments, thus avoiding many of the problems inherent in the currently available DNA extraction protocols (basically the use of expensive and/or toxic chemical reagents), and may be useful for daily clinical routine. The PCR-based system described here uses a single pair of primers (SC1F and SC1R) deduced from the C. albicans-specific KER1 gene sequence. These primers amplify a 670-bp fragment of the KER1 gene. All the clinical C. albicans isolates generated the expected 670-bp amplicon. Other non-albicans Candida species, including the azole-resistant C. krusei and C. glabrata, and the very closely related C. dubliniensis, failed to amplify any DNA fragment. The PCR results reported here suggest that amplification with SC1F and SC1R primers is species specific and, consequently, may be useful for specifically identifying C. albicans strains.     

Cleavage of human big endothelin-1 by Candida albicans aspartic proteinase

Abstract A Candida albicans aspartic proteinase (CAP), one of the secretory proteinases of Candida albicans , is thought to be a possible virulence factor in Candida albicans infection. Whereas endothelin-1 is found as an endothelium-derived strong vasoconstrictive peptide, it is known to have a role in the maintenance of vascular homeostasis and tissue survival. Endothelin-1 is generated from a precursor form of endothelin-1, the so-called big endothelin-1. It has recently been reported that cathepsin D, E and pepsin, which are aspartic proteinases, convert big endothelin-1 to endothelin-1. In this study, the relationship between CAP and big endothelin-1 was studied. High performance liquid chromatography analysis revealed that big endothelin-1 was cleaved into several amino acid sites by CAP, but endothelin-1 was not converted from big endothelin-1. CAP cleaved big endothelin-1 at different sites when compared with that of other known aspartic proteinases, and it suppressed endothelin-1 production through the degradation of big endothelin-1. CAP may break homeostatic mechanism of endothelin-1 in Candida albicans infectious lesion.     

Promoter regulation in Candida albicans and related species

Regulation of gene expression has been studied extensively in Saccharomyces cerevisiae and Schizosaccharomyces pombe . Some, but by far not all, of the findings are also applicable to Candida albicans , an important ascomycete fungal pathogen of humans. Areas of research in C. albicans include the influence of key signal transduction cascades on morphology, and the response to host-generated influences, such as host immune effector cells, blood, pH or elevated carbon dioxide. The resistance to antifungal agents and response to stress are also well researched. Conditional gene expression and reporter genes adapted to the codon usage of C. albicans are now widely used in C. albicans . Here we present a comprehensive overview of the current techniques used to investigate regulation mechanisms for promoters in C. albicans and other Candida species. In addition, we discuss reporter genes used for the study of gene expression.     

Candida albicans RHO1 is required for cell viability in vitro and in vivo

In Saccharomyces cerevisiae, Rho1p plays an important role in cell wall integrity by regulating beta-1,3-glucan synthase, Pkc1p and the actin cytoskeleton. To determine the physiological role of Rho1p in the dimorphic fungus Candida albicans, the major human fungal pathogen, we constructed mutants that conditionally express Rho1p from the glucose-repressible phosphoenolpyruvate carboxykinase promoter (pPCK1). We examined the growth of these cells in a range of conditions. Depletion of Rho1p from yeast cells resulted in cell death, lysis, and aggregation. The Rho1p conditional mutant was inviable on 10% serum indicating that Rho1p was also required for hyphal viability. Furthermore, in a mouse model of systemic candidiasis, strains dependent on pPCK1-driven RHO1 expression failed to colonise the kidneys and establish disease, suggesting that the level of glucose in serum was sufficient to repress the pPCK1 and that Rho1p-depleted strains were inviable within the host. Therefore, Rho1p is essential for the viability of C. albicans in vitro and in vivo.     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

Azole sensitivity in Candida albicans

Abstract In the present study, we assessed the influence of three culture media on the susceptibility 'in vitro' of twenty four clinical strains belonging to Candida albicans against three imidazole-derivatives and also, we investigated the situation of azole sensitivity in three of these strains.     

Antagonistic interplay of Swi1 and Tup1 on filamentous growth of Candida albicans

Candida albicans is a polymorphic human opportunistic pathogen in which the Swi-Snf complex functions as an activator whereas Tup1 acts as a general repressor during the yeast-hyphae transition. In Saccharomyces cerevisiae, the interplay between the Swi-Snf complex and the Tup1-Ssn6 repressive complex regulates the balance between active and repressed chromatin structures of a number of genes. To study the interplay between Candida albicans Swi1 and Tup1 and their effects on morphogenesis, we analyzed phenotypes of swi1/swi1, tup1/tup1 and swi1/swi1 tup1/tup1 mutants under various growth conditions. The swi1/swi1 mutant failed to form true hyphae, whereas the tup1/tup1 mutant exhibited constitutive filamentous growth. Deletion of SWI1 in the tup1/tup1 mutant completely blocked hyphal growth under all the conditions examined. Under aerobic conditions, the swi1/swi1 tup1/tup1 mutant most resembled the swi1/swi1 mutant in phenotype, actin polarization and gene expression pattern. In invaded agar, the double mutant showed similar phenotypes as the swi1/swi1 mutant, while under embedded conditions, it grew as a pseudohypha-like form different from that of the wild-type strain, swi1/swi1 or tup1/tup1 mutants. These results suggest that Swi1 may play a dominant role by antagonizing the repressive effect of the Tup1 on hyphal development in C. albicans.     

白念珠菌SPE1基因高表达菌株构建及鉴定

目的构建白念珠菌SPEl基因高表达菌株。方法将白念珠菌．SPEl基因的ORF置于高表达质粒载体pCaE—xP的MErF3启动子后面,构建pCaEXP—SPEJ的高表达质粒,然后采用醋酸锂转染法将高表达质粒转染白念珠菌RMl000中,在SD—ura’met—cys-选择性固体培养基上筛选阳性克隆,抽取基因组进行PCR验证,将验证为阳性转染子的菌落采用RealTimeRT．PCR方法进行SPE1基因转录水平的表达验证。结果通过酶切鉴定pCaEXP—SPEl高表达质粒构建正确;通过PCR验证表明SPEl基因整合到亲本菌中的RPl0位点;通过RealTimeRT—PCR方法筛选出sPEJ基因在转录水平高表达的菌株。结论利用高表达质粒载体pCaEXP通过基因同源重组等方法正确构建SPEl基因高表达的白念珠菌。